DESCRIPTION:Regulation of actin dynamics is a fundamental requirement in all eukaryotes. Endocytosis, cell motility and cell morphogenesis all rely on rapid remodeling of actin networks, which must be temporally and spatially regulated. The goal of our research is to define the molecular mechanisms governing actin assembly and organization. We are exploring this issue using the budding yeast Saccharomyces cerevisiae as a model organism to study conserved components of the cytoskeleton. Previously, we identified and characterized the yeast homologue of coronin, a ubiquitous actin associated protein with poorly defined cellular functions. We now demonstrate that coronin associates in vivo with the aboutctin- aboute1ated protein (Arp) 2/3 complex, a central nucleator of actin filament assembly and branching in eukaryotic cells. Further, purified coronin binds directly to the Arp2/3 complex and promotes actin filament debranching and turnover. This activity is consistent with our previously observed genetic interactions between coronin and cofilin and may define a conserved cellular function of coronin. The Arp2/3 complex has 7 different sub-units and is regulated by a large number of activators and other factors, including the Wiskott-Aldrich Syndrome (WAS) protein. We have identified a new activator of the Arp2/3 complex, Abplp. Our preliminary results suggest that WASp and Abplp activate Arp2/3 complex by distinct mechanisms, and we will directly test this hypothesis. Further, we have in vivo evidence that there are two distinct pathways in cells for regulating Arp2/3 complex, one that is WASp-dependent and one that is Abplp dependent. We will take a combined genetic and biochemical approach to dissect these in vivo pathways and to uncouple the activities and interactions of the Arp2/3 complex. This will allow us to determine how Arp2/3 complex activities are regulated in different cellular processes. The specific aims are: (1) Characterize the coroninArp2/3 complex activity and test its functional significance in vivo, (2) Dissect pathways for differential regulation of Arp2/3 complex functions in vivo and define their underlying biochemical basis, (3) Uncouple Arp2/3 complex activities and in vivo functions using a large collection of arc35 mutant alleles, and (4) Define the mechanisms 01 Arp2/3 complex activation by WASp and Abplp.